Targeting primary cilia to treat glaucoma

ABSTRACT

Methods for reducing intraocular pressure and treating glaucoma are disclosed. The methods include administering to an individual a composition comprising a TRPV4 agonist.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/941,601 filed on Feb. 19, 2014, which is hereby incorporated byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under EY022058 awardedby National Institutes of Health. The Government has certain rights inthe invention.

STATEMENT IN SUPPORT FOR FILING A SEQUENCE LISTING

A paper copy of the Sequence Listing and a computer readable form of theSequence Listing containing the file named “IURTC 2014-099-02_ST25.txt”,which is 730 bytes in size (as measured in MICROSOFT WINDOWS® EXPLORER),are provided herein and are herein incorporated by reference. ThisSequence Listing consists of SEQ ID NOs:1-3.

BACKGROUND OF THE DISCLOSURE

The present disclosure relates generally to treating diseases anddisorders resulting from defects in cilia formation, cilia maintenanceand cilia function. More particularly, the present disclosure relates tomethods for reducing eye pressure, methods for treating ciliopathies andmethods for treating glaucoma by administering transient receptorpotential cation channel subfamily V member 4 (TRVP4) agonists.

The primary cilium is an evolutionarily conserved subcellular structurethat protrudes from many post-mitotic eukaryotic cells. In response tochanges in the extracellular environment, primary cilia coordinatesignaling cascades that control cell differentiation, growth andfunction. A highly specialized extension of the plasma membrane, theciliary membrane is enriched with many signaling proteins, includingTransient Receptor Potential (TRP) channels. Upon extracellularstimulation, TRP channels initiate signal transduction cascades byinducing Ca²⁺ flow. Phosphoinositides within the ciliary membrane areessential secondary messengers for ciliary function, potentially throughmodulation of the activities of TRP channels.

Transient receptor potential cation channel subfamily V member 4 (TRPV4)is a member of the OSM9-like transient receptor potential channel(OTRPC) subfamily that in humans is encoded by the TRPV4 gene. TRPV4protein is a Ca²⁺-permeable, nonselective cation channel that is thoughtto be involved in the regulation of systemic osmotic pressure. TRPV4also functions as a ciliary mechanosensory channel. Mutations in theTRPV4 gene have been associated with disorders including brachyolmiatype 3, congenital distal spinal muscular atrophy, scapuloperonealspinal muscular atrophy and subtype 2C of Charcot-Marie-Tooth disease. Anumber of TRPV4 agonists and antagonists have been identified including,for example, the antagonist Ruthenium Red, the agonist 4aPDD, theselective antagonist RN-1734, the agonist GSK1016790A and the antagonistHC-067047.

Defects in cilia formation or maintenance underlie a wide range of humandiseases, including retinitis pigmentosa, renal cysts, polydactyly, anddevelopmental delays, which are collectively called ciliopathies. It hasbeen discovered that OCRL, an inositol polyphosphate 5-phosphataseimplicated in Oculocerebrorenal syndrome of Lowe (Lowe syndrome), a rareX-linked recessive disorder that presents in males with bilateralcataracts and glaucoma, as well as renal failure, muscular hypotonia,and mental retardation, regulates cilia biogenesis. OCRL substratesinclude phosphatidylinositol-4,5-bisphophatase [PI(4,5)P₂] andphosphatidylinositol-3,4,5-triphosphate [PI(3,4,5)P₃]. Decreased5-phosphatase activity is demonstrated in fibroblasts from Lowepatients, as well as a two- to threefold elevated ratio ofPI(4,5)P₂:PI(4)P.

Mechanosensation of pressure underlies a number of important humandiseases including the development of hypertension and glaucoma. In thekidney epithelium, ciliary proteins polycystins (PC1/2) have been shownto be important for flow-dependent calcium flux. In the lining of theventricles of the brain, cerebrospinal fluid is regulated by cilia.Similar to the kidney, the eye is an enclosed organ with sensitivehomeostatic regulation of fluid production and egress. Defectivesensation of pressure may result in imbalance of aqueous humor,resulting in elevated intraocular pressure. Low levels of eye pressureresult in structural changes of the retina and poor vision, whileelevated eye pressure may damage the optical nerve. Glaucoma is an opticneuropathy associated with elevated intraocular pressure and is aleading cause of irreversible blindness in the world.

Trabecular meshwork cells are responsible for the drainage of themajority of aqueous fluid. Dysfunction of the trabecular outflow leadsto elevated intraocular pressure, which in susceptible individuals,results in the death of retinal ganglion cells that leads toirreversible vision loss. However, the molecular events whereby elevatedpressure results in aberrant mechanosensory signaling that lead tovisual loss are poorly understood. As disclosed herein, trabecularmeshwork cells of the eye have primary cilia that are responsive topressure changes.

Consistent with the central role of increased pressure in the pathologyof glaucoma, the only proven treatment is lowering of pressure. Fiveclasses of medications are available for treating glaucoma, whichinclude beta blockers, alpha adrenergic agonists, carbonic anhydraseinhibitors, cholinergic agonists and prostaglandin analogs. Manypatients become intolerant of the side effects of these medications. Itis also recognized that these medications cease to lower pressure aftera number of years. As patients become intolerant of medications and asthe medications lose their effectiveness, surgical intervention isrequired to lower pressure.

Accordingly, there exists a need to develop alternative treatments forlowering eye pressure.

BRIEF DESCRIPTION OF THE DISCLOSURE

The present disclosure relates generally to treating diseases anddisorders resulting from defects in cilia formation, cilia maintenanceand cilia function. More particularly, the present disclosure relates tomethods for reducing eye pressure, methods for treating ciliopathies andmethods for treating glaucoma by administering transient receptorpotential cation channel subfamily V member 4 (TRVP4) agonists.

In one aspect, the present disclosure is directed to a method forreducing intraocular pressure in an individual in need thereof. Themethod includes administering a TRPV4 agonist to the individual.

In another aspect, the present disclosure is directed to a method fortreating glaucoma in an individual in need thereof. The method includesadministering a TRPV4 agonist to the individual.

In another aspect, the present disclosure is directed to treating aciliopathy in an individual in need thereof. The method includesadministering a TRPV4 agonist to the individual.

In another aspect, the present disclosure is directed to apharmaceutical formulation for reducing intraocular pressure including:from about 50 ng/g body weight to about 500 ng/g body weight a TRPV4agonist.

In accordance with the present disclosure, methods have been discoveredthat surprisingly allow for the treatment for intraocular pressure and,in particular, glaucoma. The present disclosure has a broad andsignificant impact, as it allows for treating glaucoma and dysregulationof eye pressure that can lead to vision loss and blindness.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The disclosure will be better understood, and features, aspects andadvantages other than those set forth above will become apparent whenconsideration is given to the following detailed description thereof.Such detailed description makes reference to the following drawings,wherein:

FIG. 1 is a schematic illustration of aqueous humor outflow in the eye,as discussed in Example 1.

FIG. 2A is an immunofluorescent micrograph of the trabecular meshworkremoved from human donor eyes and immunostained with anti-Arl13b (red),anti-acetylated α-tubulin (AcTub, red), anti-γ-tubulin (γ-Tub, green)and DAPI (blue), as discussed in Example 1. Scale bar=5 μm.

FIG. 2B is an immunofluorescent micrograph of surgical trabecularmeshwork specimens obtained during glaucoma surgery and immunostainedwith anti-Arl13b (red), anti-acetylated α-tubulin (AcTub, red),anti-γ-tubulin (γ-Tub, green) and DAPI (blue), as discussed inExample 1. Scale bar=5 μm.

FIG. 2C are transmission electronmicrographs of trabecular meshworksections of human eyes showing basal body and axonemal structures(indicated with arrows) and the cell nucleus (N), as discussed inExample 1. Scale bar=500 nm.

FIG. 2D is a transmission electronmicrograph of trabecular meshworksection of a human eye showing basal body and axonemal structures(indicated with arrows), as discussed in Example 1. Scale bar=500 nm.

FIG. 2E depicts characterization of human TM tissue. Human trabeculartissues were stained with anti-myocilin antibody (green) and DAPI(blue). Phase-contrast and immunofluorescence images were obtained.(Scale bar, 10 μm).

FIG. 2F is a fluorescence micrograph of trabecular meshwork cilia frommurine eyes stained with anti-Arl13b (red) anti-γ-tubulin (γ-Tub, green)and DAPI (blue), as discussed in Example 1. Scale bar=5 μm.

FIG. 2G is a high magnification scanning electron micrograph showingtrabecular meshwork cilia from porcine eyes, as discussed in Example 1.

FIG. 2H is a low magnification scanning electron micrograph showingtrabecular meshwork cilia from porcine eyes, as discussed in Example 1.Scale bar=5 μm.

FIGS. 2I and 2J are fluorescence micrographs of trabecular meshworkcilia from bovine eyes stained with anti-Arl13b (AcTub, red)anti-α-tubulin (red), anti-γ-tubulin and (γ-Tub, green) and DAPI (blue),as discussed in Example 1. Scale bar=10 m.

FIG. 2K are immunofluorescent micrographs of HTM cells serum-starved toinduce ciliogenesis and immunostained with anti-Arl3b (red),anti-acetylated α-tubulin (AcTub, red), anti-IFT-88 (green), anti-IFT43(green), anti-TFT57 (green), anti-adenylate cyclase III (AC III, green),anti-pericentrin (green), anti-γ-tubulin (γ-Tub, green) and DAPI (blue),as discussed in Example 1. Insets show higher magnification of cilia.Scale bar=5 μm.

FIG. 3A are fluorescence micrographs showing cilia formation inserum-starved HTM cells for 24 hours, 48 hours and 72 hours stained withanti-acetylated α-tubulin (red) and DAPI (blue), as discussed inExample 1. Scale bar=10 μm.

FIG. 3B is a graph illustrating cilia length measurements inserum-starved HTM cells for 24 hours, 48 hours and 72 hours, asdiscussed in Example 1. Error bars represent standard deviation (SD).N>50 cilia, three independent experiments, ANOVA, *, p<0.001.

FIG. 3C is a graph illustrating ciliation rate in serum-starved HTMcells for 24 hours, 48 hours and 72 hours, as discussed in Example 1.Error bars represent standard deviation (SD). N>50 cilia, threeindependent experiments, ANOVA, *, p<0.001.

FIG. 4A are fluorescent micrographs of serum-starved HTM cells that werecultured in atmospheric (control, 0 mmHg) and 50 mmHg pressure andimmunostained with anti-acetylated α-tubulin (AcTub, red), and anti-γtubulin (γ-Tub, green). Insets show higher magnification of cilia, asdiscussed in Example 2. Scale bar=5 μm.

FIG. 4B is a graphical illustration depicting measurements of cilialength of serum-starved HTM cells that were cultured in atmospheric(control, 0 mmHg), 30 mmHg pressure and 50 mmHg pressure from threedifferent experiments, as discussed in Example 2. Error bars representstandard deviation. N>50 cilia; paired t-test *, p value<0.05.

FIG. 4C is a graphical illustration depicting measurements of cilialength of serum-starved HTM cells that were cultured in 50 mmHg pressurefor 0 hour, 1 hour and 3 hours and immunostained with anti-acetylatedα-tubulin (AcTub, red) to measure cilia length, as discussed in Example2. Error bars represent standard deviation. N>50 cilia; unpaired t-test*, **p value<0.05, ns—not significant.

FIG. 4D is a graphical illustration depicting mean mRNA levels of TNFα,Gli1 and TGFβ measured by RT-PCR in serum-starved HTM cells treated withsaline (control), saline plus 50 mmHg pressure, chlorohydrate (+CH Hy),and chlorohydrate plus 50 mmHg pressure (+CH Hy+50 mmHg) for 3 hoursfrom three independent experiments, as discussed in Example 2. Errorbars represent standard deviation, * t-test p value<0.05, ns—notsignificant.

FIG. 4E is a graph illustrating mean relative expression of TNFαmRNA andIL-33 mRNA in serum-starved HTM cells and placed under 50 mmHg pressurefor 0, 10, 20, 40, 60 and 80 minutes, as discussed in Example 2. Mean ofthree independent experiments is shown. Error bars represent SD.

FIG. 4F is a graphical illustration depicting mean mRNA levels of TNFα,Gli1 and TGFβ measured by RT-PCR during cilia formation in serum-starvedHTM cells (control) and serum-starved HTM cells (Rab8 knockdown)followed by treatment with saline and saline plus 50 mmHg pressure for 3hours from three independent experiments, as discussed in Example 2.Error bars represent standard deviation, * p value<0.05, ns—notsignificant.

FIG. 4G is an immunoblot for Rab8 level and β-Actin (loading control)level from control KD HTM cell lysates and Rab8 KD HTM cell lysates, asdiscussed in Example 2.

FIG. 4H is an immunoblot for OCRL level and β-Actin (loading control)level from control KD HTM cell lysates and OCRL KD HTM cell lysates, asdiscussed in Example 2.

FIG. 5 are fluorescence micrographs of representative images ofimmunostaining with antibodies for IFT88 (green), acetylated α-tubulin(red), and DAPI (blue), showing the loss of IFT88 in the distal tip ofcilia under elevated pressure conditions, as discussed in Example 3.(Scale bar, 10 μm.)

FIG. 6 is a graph showing altered distribution of IFT88 with elevatedpressure. HTM cells were serum-starved to induce ciliogenesis, followedby treatment with or without elevated pressure (50 mmHg).

FIG. 7 is a photograph of the optic nerve of a patient with congenitalglaucoma and cataracts depicting glaucomatous optic nerve cupping (asindicated by dashed line), as discussed in Example 4.

FIG. 8A is a graphical illustration showing the 1661 A>C DNA mutation(D499A amino acid mutation) identified by sequencing PCR products fromkeratinocytes of a Lowe 3 affected patient, as discussed in Example 4.

FIG. 8B is an illustration depicting the protein structure of the OCRL5-phosphatase domain showing the site of mutation, as discussed inExample 4.

FIG. 8C are fluorescence micrographs of cilia formation in serum-starvedkeratinocytes from normal control (NHKC), keratinocytes from a Lowesyndrome patient (Lowe 3) and keratinocytes from a patient transducedwith WT-GFP-OCRL (Lowe 3+WT) that were stained with anti-acetylatedα-tubulin, as discussed in Example 4. Scale bar=10 μm.

FIG. 8D is a graph illustrating cilia lengths from three independentexperiments of cells described in FIG. 3C, as described in Example 3.N=50 cilia, ANOVA, P<0.001.

FIG. 8E are fluorescence micrographs of serum-starved HTM cellstransfected with GFP-WT OCRL and GFP-D499A-OCRL that were stained withanti-acetylated α-tubulin, as discussed in Example 4. Scale bar=5 μm.

FIG. 8F is a graph illustrating cilia length measurements fromserum-starved HTM cells transfected with GFP-WT OCRL and GFP-D499A-OCRL,as discussed in Example 4. Error bars represent SD. N>50 cilia, threeindependent experiments, unpaired t-test.

FIG. 8G is a graph illustrating ciliation percent measurements fromserum-starved HTM cells transfected with GFP-WT OCRL and GFP-D499A-OCRL,as discussed in Example 4. Error bars represent SD. N>50 cilia, threeindependent experiments, unpaired t-test.

FIG. 8H are fluorescence micrographs of serum-starved control HTM(Control KD), OCRL KD HTM (OCRL KD) and OCRL-WT expressing (OCRL KD+WTrescue) OCRL KD HTM cells incubated with or without 50 mmHg pressure for3 hours, as discussed in Example 4. Representative photomicrographsshowed short cilia in HTM cells with 50 mmHg and OCRL KD HTM cells.Scale bar 5 μm.

FIG. 8I is a graph illustrating cilia length measurements fromserum-starved control HTM (Control KD), OCRL KD HTM (OCRL KD) andOCRL-WT expressing (OCRL KD+WT rescue) OCRL KD HTM cells incubated withor without 50 mmHg pressure for 3 hours, as discussed in Example 4.Error bars represent SD. N>50 cilia, three independent experiments,unpaired t-test.

FIG. 8J is a graph illustrating pressure induced TNFα, TGFβ and Gli1mRNA levels in serum-starved control HTM (HTM), OCRL KD HTM (OCRL KD)and OCRL-WT expressing OCRL KD HTM (OCRL KD+WT rescue) cells incubatedwith or without 50 mmHg pressure for 3 hours followed by RT-PCR, asdiscussed in Example 4. Error bars represent standard deviation, pairedt-test, P<0.001.

FIG. 8K is a graph illustrating pressure induced TGFβ mRNA level inserum-starved NHF558 fibroblasts, Lowe 1 or Lowe 2 fibroblasts andOCRL-WT expressing Lowe 1 (Lowe 1+WT rescue) or Lowe 2 (Lowe 2+WTrescue) fibroblasts incubated with or without 50 mmHg pressure for 3 hrfollowed by RT-PCR, as discussed in Example 4. Error bars representstandard deviation, paired t-test, P<0.001.

FIG. 9A are fluorescence micrographs of serum-starved HTM cells stainedwith anti-TRPV4 (green), anti-acetylated α-tubulin (red) and DAPI(blue), as discussed in Example 5. Scale bar=5 μm.

FIG. 9B is an immunoblot for OCRL and TRPV4 levels (β-Actin loadingcontrol) from NHF cell lysates, Lowe 1 cell lysates, Lowe 2 celllysates, and HTM cell lysates, as discussed in Example 5.

FIG. 9C is an immunoblot for OCRL and TRPV4 levels (β-Actin loadingcontrol) from NHKC cell lysates and Lowe 3 cell lysates, as discussed inExample 5.

FIG. 9D is an immunoblot (IB) performed after immunoprecipitation ofFLAG detecting OCRL and FLAG-TRPV4 in HEK293 cells transfected withFLAG-TRPV4, GFP-OCRL and GFP alone, as discussed in Example 5.

FIG. 9E are fluorescence micrographs of serum-starved HTM cellstransfected with FLAG-TRPV4, GFP-OCRL and GFP-D499A-OCRL stained withanti-acetylated α-tubulin (red) and anti-FLAG, as discussed in Example5. OCRL and D499A are shown in green; FLAG-TRPV4 and DAPI are shown inblue. Scale bar=5 μm.

FIG. 9F is an immunoblot (IB) performed after immunoprecipitation ofFLAG detecting OCRL and FLAG-TRPV4 in HEK293 cells transfected withFLAG-TRPV4, GFP-OCRL and GFP-D499A-OCRL, as discussed in Example 5.

FIG. 10A is a graph depicting that OCRL D499A mutant exhibited decreasedPI(4,5)P2 5-phosphatase activity. HTM cells transfected with FLAG-alone,FLAG-OCRL-WT, or FLAG-OCRL-D499A, immunoprecipitated with anti-FLAGbeads and subjected to PI(4,5)P2 5-phosphatase assay. Enzymatic activityis indicated as a ratio with the wild-type OCRL; average of threeindependent experiments is shown. Error bars represent SD. Paired ttest, *P<0.05

FIGS. 10B and 10C depict that OCRL-WT and D499A mutant exhibited similarhalf-life for protein stability. The levels of GFP-OCRL-WT.GFP-OCRL-D499A, and endogenous α-actin were detected by immunoblot oflysates from HEK293T cells transfected with GFP-OCRL-WT orGFP-OCRL-D499A following treatment for the indicated times with CHX (200μg/mL). A graph is shown of the ratios of GFP-OCRL to α-actin and theresulting half-lives (t1/2) in cells expressing GFP-OCRL-WT orGFP-OCRL-D499A (FIG. 10C).

FIG. 11A are micrographs of HTM (Lowe 1) cells and NHF cells stainedwith Fura2 to visualize calcium flow, as discussed in Example 6. Scalebar=10 μm.

FIG. 11B is a graph illustrating fluorescence ratio of F340/F380 levelsas an indication of calcium mobilization in NHF, Lowe 1 and Lowe 2 cellsstained with Fura2 followed by treatment with 0.1 M of the TRPV4 agonistGSK1016790A, as discussed in Example 6. Error bars represent standarddeviation.

FIG. 11C is a graph illustrating intraocular pressure in upk^(−/−) ratsthat were sham treated, treated with the TRPV4 agonist GGSK 1016790A,and treated with the TRPV4 antagonist HC 067047, as discussed in Example6. Error bars represent standard deviation, unpaired t-test, *, p<0.001.

FIG. 12A is a graph illustrating defective TRPV4-mediated calciumsignaling in HTM cells under elevated pressure, as discussed in Example6.

FIG. 12B is a graph illustrating TRPV4-mediated calcium signaling in HTMcells, as discussed in Example 6.

FIG. 12C is a graph illustrating defective TRPV4-mediated calcium fluxin Lowe syndrome patient cells, as discussed in Example 6.

FIG. 12D is a graph illustrating that TRPV4 agonist, but not anantagonist, lowers IOP in mouse, as discussed in Example 6.

FIG. 12E is a graph illustrating that TRPV4: mice exhibited higher IOPthan TRPV4+/+ mice, as discussed in Example 6.

FIG. 12F is a graph illustrating shortened cilia in TM cells ofTRPV4^(−/−), as discussed in Example 6.

FIG. 12G are fluorescence micrographs of shortened cilia in TM cells ofTRPV4^(−/−), as discussed in Example 6.

FIGS. 13A & 13B are graphs illustrating the effect of TRPV4 agonists andantagonists on IOP lowering effect in C57BI/6 mice, as discussed inExample 7.

FIGS. 14A & 14B are graphs illustrating the time-dependent loweringeffect of TRPV4 agonists, as discussed in Example 8.

FIG. 15 are fluorescence micrographs illustrating the expression ofTRPV4 in human TM cells, as discussed in Example 9.

FIG. 16 is an illustration of a model for OCRL and TRPV4 in the cilia oftrabecular meshwork cells.

While the disclosure is susceptible to various modifications andalternative forms, specific embodiments thereof have been shown by wayof example in the drawings and are herein described below in detail. Itshould be understood, however, that the description of specificembodiments is not intended to limit the disclosure to cover allmodifications, equivalents and alternatives falling within the spiritand scope of the disclosure as defined by the appended claims.

DETAILED DESCRIPTION OF THE DISCLOSURE

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure belongs. Although any methods andmaterials similar to or equivalent to those described herein can be usedin the practice or testing of the present disclosure, the preferredmethods and materials are described below.

Although some forms of ciliopathies present with disease states of fluiddysregulation, including hydrocephalus, it has been previously unknownif elevated eye pressure may be caused by ciliary disease. Glaucoma isan optic neuropathy associated with elevated intraocular pressure (IOP)and is a leading cause of irreversible blindness in the world.Consistent with the central role of increased pressure in the pathologyof glaucoma, the only proven treatment for glaucoma is to lower eyepressure. Trabecular meshwork (TM) cells are responsible for thedrainage of the majority of aqueous fluid, and dysfunction of thetrabecular outflow leads to elevated IOP. In susceptible individuals(e.g., Lowe patients), this results in the death of retinal ganglioncells, causing irreversible vision loss. However, prior to the presentdisclosure, the molecular events whereby elevated pressure results inaberrant mechanosensory signaling that leads to vision loss are poorlyunderstood.

In accordance with the present disclosure, treatments have beendiscovered that surprisingly allow for treating diseases and disordersresulting from defects in cilia formation, cilia maintenance and ciliafunction. Elevated eye pressure can lead to vision loss, blindness andglaucoma. Advantageously, the methods of the present disclosure allowfor lowering intraocular pressure as treatments for elevated eyepressure associated with vision loss, blindness and glaucoma, as well astreatments for ciliopathies.

In one aspect, the present disclosure is directed to a method forreducing intraocular pressure in an individual in need thereof. Themethod includes administering a TRPV4 agonist to the individual.

In another aspect, the present disclosure is directed to a method fortreating glaucoma in an individual in need thereof. The method includesadministering a TRPV4 agonist to the individual.

In another aspect, the present disclosure is directed to treating aciliopathy in an individual in need thereof. The method includesadministering a TRPV4 agonist to the individual. The ciliopathy can be,for example, retinitis pigmentosa, renal cysts, polydactyly, anddevelopmental delays.

As used herein “transient receptor potential vanilloid 4 agonist” or“TRPV4 agonist” refers to any compound capable of activating orenhancing the biological activities of a TRPV4 channel receptor.Suitable agonists to TRPV4 channel receptors can be, for example,compounds included in the class of 3-oxohexahydro-1H-azepin, azepine andacyclic 1,3-diamine and derivatives of these compounds. Suitable TRPV4agonists can be, for example, GSK1016790A(N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide);N-{(1S)-1-[({(4R)-1-[(4-chlorophenyl)sulfonyl]-3-oxohexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl}-1-benzothiophene-2-carboxamide;N-{(1S)-1-[({(4R)-1-[(4-fluorophenyl)sulfonyl]-3-oxohexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl)-1-benzothiophene-2-carboxamide;N-{(1S)-1-[(((4R)-1-[(2-cyanophenyl)sulfonyl]-3-oxohexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl}-1-methyl-1H-indole-2-carboxamide;N-{(S1)-1-[({(4R)-1-[(2-cyanophenyl)sulfonyl]hexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl}-1-methyl-1H-indole-2-carboxamide;N-{(1S)-1-[({3-[[(cyanophenyl)sulfonyl](methyl)amino]propyl}amino)carbonyl]-3-methylbutyl}-1-benzothiophene-2-carboxamide;andN-{(1S)-1-[({3-[[(2,4-dichlorophenyl)sulfonyl](methyl)amino]propyl}amino)carbonyl]-3-methylbutyl}-1-benzothiophene-2-carboxamide.Other suitable TRVP4 agonists are disclosed in US 2007/0259856 andInternational Patent Applications WO 2000/03687, WO 2001/095911, WO2002/017924, and WO 2006/029209, which are incorporated by referenceherein to the extent they are consistent herewith.

Suitable amounts of the TRPV4 agonist can have, for example, an EC50value for TRPV4 channel receptor of about 1.0 μM. Particularly suitableamounts of the TRPV4 agonist can have, for example, an EC50 value forTRPV4 channel receptor of less than 1.0 μM. In another aspect, theagonist can have, for example, an EC50 value for TRPV4 channel receptorof about 10 nM. Particularly suitable amounts of the TRPV4 agonist canhave, for example, an EC50 value for TRPV4 channel receptor of less than10 nM. In another aspect, the agonist has an EC50 value for TRPV4channel receptor of about 1.0 μM as measured by calcium influx inisolated trabecular meshwork cells, human trabecular meshwork cells,normal human fibroblasts, Lowe syndrome patient keratinocytes, or Lowesyndrome patient fibroblasts. In particular, the agonist has an EC50value for TRPV4 channel receptor of less than 1.0 μM as measured bycalcium influx in isolated trabecular meshwork cells, human trabecularmeshwork cells, normal human fibroblasts, Lowe syndrome patientkeratinocytes, or Lowe syndrome patient fibroblasts.

A particularly suitable dosage can be from about 50 ng/g body weight toabout 500 ng/g body weight, including from about 50 ng/g body weight toabout 250 ng/g body weight, including from about 50 ng/g body weight toabout 100 ng/g body weight, and including about 50 ng/g body weight.Suitable dosage of a TRPV4 channel receptor agonist of the presentdisclosure will depend upon a number of factors including, for example,age and weight of an individual, at least one precise conditionrequiring treatment, severity of a condition, nature of a formulation,route of administration and combinations thereof. Ultimately, a suitabledosage can be readily determined by one skilled in the art such as, forexample, a physician, a veterinarian, a scientist, and other medical andresearch professionals. For example, one skilled in the art can beginwith a low dosage that can be increased until reaching the desiredtreatment outcome or result. Alternatively, one skilled in the art canbegin with a high dosage that can be decreased until reaching a minimumdosage needed to achieve the desired treatment outcome or result.

As used herein and as is understood in the art “EC50” or “effectiveconcentration 50%” refers to the molar concentration of an agonist thatproduces 50% of the maximum possible stimulatory response for thatagonist. The maximum stimulatory response for each agonist can bedetermined experimentally by measuring the magnitude of the desiredbiological response elicited by increasing concentrations of agonistuntil a plateau is achieved.

As used herein and as is understood in the art “IC50” or “inhibitoryconcentration 50%” refers to the molar concentration of a compound(agonist, antagonist, or inhibitor) that produces 50% of the maximumpossible inhibitory response for that compound. The maximum inhibitoryresponse for each compound can be determined experimentally by measuringthe extent of inhibition of the desired biological response elicited byincreasing concentrations of agonist until a plateau is achieved.

Any suitable method known to those skilled in the art can be used foradministering the TRPV4 agonist. Suitable methods for administering theTRPV4 agonist can be, for example, topically, periocularly,intraocularly, by intraocular injection and other types ofadministration methods known to those skilled in the art.

The TRPV4 agonist of the disclosure can be administered as apharmaceutical composition comprising the TRPV4 agonist of interest incombination with one or more pharmaceutically acceptable carriers. Asused herein, the phrase “pharmaceutically acceptable” refers to thoseligands, materials, formulations, and/or dosage forms which are, withinthe scope of sound medical judgment, suitable for use in contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. The phrase“pharmaceutically acceptable carrier”, as used herein, refers to apharmaceutically acceptable material, formulation or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the active compound fromone organ or portion of the body, to another organ or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other components of the composition (e.g., TRPV4 agonist) andnot injurious to the individual. Lyophilized compositions, which may bereconstituted and administered, are also within the scope of the presentdisclosure.

Pharmaceutically acceptable carriers may be, for example, excipients,vehicles, diluents, and combinations thereof. For example, where thecompositions are to be administered by the ophthalmic mucous membraneroute, they may be formulated as eye drops or eye ointments. Thesecompositions can be prepared by conventional means, and, if desired, theactive compound (i.e., TRPV4 agonist) may be mixed with any conventionaladditive, such as an excipient, a binder, a disintegrating agent, alubricant, a corrigent, a solubilizing agent, a suspension aid, anemulsifying agent, a coating agent, or combinations thereof.

It should be understood that the pharmaceutical compositions of thepresent disclosure can further include additional known therapeuticagents, drugs, modifications of the synthetic compounds into prodrugs,and the like for alleviating, mediating, preventing, and treating thediseases, disorders, and conditions described herein.

The pharmaceutical compositions including the TRPV4 agonist andpharmaceutical carriers used in the methods of the present disclosurecan be administered to a subset of individuals in need. As used herein,an “individual in need” refers to an individual at risk for or havingelevated intraocular pressure. Additionally, an “individual in need” isalso used herein to refer to an individual at risk for or diagnosed by amedical professional as having glaucoma. As such, in some embodiments,the methods disclosed herein are directed to a subset of the generalpopulation such that, in these embodiments, not all of the generalpopulation may benefit from the methods. Based on the foregoing, becausesome of the method embodiments of the present disclosure are directed tospecific subsets or subclasses of identified individuals (that is, thesubset or subclass of individuals “in need” of assistance in addressingone or more specific conditions noted herein), not all individuals willfall within the subset or subclass of individuals as described herein.In particular the individual in need is a human. The individual in needcan also be, for example, a research animal such as, for example, anon-human primate, a mouse, a rat, a rabbit, a cow, a pig, and othertypes of research animals known to those skilled in the art.

The disclosure will be more fully understood upon consideration of thefollowing non-limiting Examples.

EXAMPLES Materials and Methods Reagents

TRPV4 agonist (GSK 1016790A), or antagonist (HC 067047) were obtainedfrom Sigma (St. Louis, Mo.) and TOCRIS (Bristol, UK), respectively.Acetylated α-tubulin, anti-β actin, anti-γ tubulin monoclonal antibodieswere purchased from Sigma (St. Louis, Mo.). Anti-TRPV4 polyclonalantibody was from Alomone Lab (Jerusalem, Israel). Anti-adenylatecyclase III antibody was from Santa Cruz Biotech (Santa Cruz, Calif.).Anti-IFT-88 polyclonal antibody was from ProteinTech (Chicago, Ill.).μ-catenin and pericentrin antibodies were obtained from PW Majerus(Washington University, St. Louis). The anti-FLAG-M2 antibody waspurchased from Agilent (Englewood, Colo.). Anti-FLAG beads were obtainedfrom Clontech (Mountain View, Calif.). Secondary antibodies AlexaFluor488 and 594 anti-mouse IgG, Cy3 anti-mouse IgG, HRP-conjugatedanti-rabbit and anti-mouse IgG were obtained from Jackson Laboratories(West Grove, Pa.). IRDye anti-mouse and anti-rabbit (680 and 800) wereobtained from Li-cor Bioscience (Lincoln, NB). Primers for Gli1, TNFα,and TGFβ and GAPDH were obtained from IDT (Coralville, Iowa).

Animals

All animal experiments followed the guidelines of the ARVO Statement forthe Use of Animals in Ophthalmic and Vision Research and were approvedby the Institutional Animal Care and Use Committee of Indiana UniversitySchool of Medicine. Wpk rats, a polycystic kidney disease model, werehoused under a 12-hour light/dark cycle with free access to water andfood (Smith et al., Nat. Genet. 2006, 38(2):191-196). For topicalanesthesia, 50 mg/ml sodium pentobarbital was injected.

Cell Culture and Tissue Isolation

The trabecular meshwork (TM) tissues were isolated from healthy humancadaver eyes by careful dissection, with Indiana University IRBapproval. Culture of TM cells was performed according to Tripathi &Tripathi (Exp. Eye Res. 1982, 35(6):611-624). The identity of trabecularmeshwork cells was established by morphology and ability to take upacetylated low-density lipoprotein and to secrete tissue plasminogenactivator. Human RPE cells (hTERT-RPE1) cell lines were previouslydescribed by Luo et al. (Hum. Mol. Genet. 2012, 21(15):3333-3344).NHF588 were obtained from D. Spandau (Indiana University) and HEK293Tcells were obtained from J. B Travers (Indiana University).

DNA Plasmid and Transfection

GFP-OCRL-WT and FLAG-OCRL-WT were obtained from Addgene (Cambridge,Mass.). Flag-TRPV4 constructs were gifts from the Lefkowitz lab (DukeUniversity, N.C.). TRPV4 cDNA was purchased from Thermo Scientific(Pittsburgh, Pa.). OCRL D499A mutant (1661 A>C) was generated usingQUIKCHANGEII® kit from Stratagene (Santa Clara, Calif.). Transfectionswere performed using LIPOFECTAMINE®2000 or Polyethylenimine (PET)according to the manufacturer's protocol (Life Technologies, GrandIsland, N.Y.).

Immunofluorescence

For immunofluorescence techniques and cilia measurement, cell cultureslides were treated with paraformaldehyde (PFA) for fixation for 10minutes at room temperature (RT) followed by permeabilization with 0.5%Triton X-100. Samples were then blocked with phosphate-buffered saline(PBS)/0.5% bovine serum albumin (BSA)/10% normal goat serum (NGS) for 30minutes at RT. Primary antibodies were applied at 4° C. overnight,followed by secondary antibodies at RT for 1 hour. Imaging for ciliameasurements was performed with a Zeiss LSM700 as described by Luo etal. (Hum. Mol. Genet. 2012. 21(15):3333-3344).

Immunoprecipitation

Co-immunoprecipitation experiments were performed as described (Kang etal., Invest. Ophthalmol. Vis. Sci. 2013, 54(4):2523-2532). Briefly,HEK293T cells were lysed in phosphate buffered saline followed byincubation with the anti-FLAG beads for 3 hours at 4° C. Theprotein-antibody complexes were then washed with lysis buffer, resolvedby SDS-PAGE and subjected to immunoblot analysis.

Immunoblot Analysis

Cell lysates were subjected to SDS-PAGE. Equal amounts of protein wererun on 10-12% gel and transferred to nitrocellulose membrane (BioRad,Hercules, Calif.). Membranes were blocked in 5% non-fat dried milk inPBS. Primary and secondary antibodies were diluted in the concentrationsdescribed in Luo et al. (Hum. Mol. Genet. 2012, 21(15):3333-3344). AnOdyssey Imaging system (Li-Cor Bioscience) was used to analyze theimmunoblots.

Quantitative Real-Time PCR

Total RNA was extracted using the RNAEASY® kit (Qiagen). Purified RNAwas quantitated with the NANODROP® 2000 (Thermo Fisher Scientific,Lafayette, Colo.). Reverse transcription of total RNA was done usingSUPERSCRIPT cDNA synthesis kit (Invitrogen) with random hexamers.Quantitative RT-PCR was performed using the RQ and ΔΔCt method on a StepOne Real-Time PCR machine (Bio-Rad Laboratories, Hercules, Calif.). Eachassay was performed in triplicate with Power SYBR green MasterMix (SABiosciences, Frederick, Md.).

Measurement of Intraocular Pressure

lntraocular Pressure (TOP) was measured with the TONOLAB tonometer TV02(Helsinki, Finland) with rats under topical anesthesia. IOP was measuredusing six valid rebound measurements of the device from the eye, wherethe mean of the middle five readings was taken for one summarymeasurement. A single set of readings was collected with the bestreproducibility indicator during the IOP measurements.

Hydrostatic Pressure Chamber

To analyze the cellular changes in the TM cells, a hydrostatic pressurechamber was developed similar to that of Mandel et al. (Invest.Ophthalmol. Vis. Sci. 2010, 51(6):3129-3138). The cells were grown in 60mm polycarbonate dishes sealed to attach to a plastic column filled with400 mL DMEM/F12 media with or without serum for cilia induction. Thecolumn was temperature controlled at 37° C. in humidified chamber. Theheight of the column was set at 67 cm for a pressure at the base of 50mmHg; 40 cm fluid column for a pressure of 30 mmHg at the base (Mandelet al., Invest. Ophthalmol. Vis. Sci. 2010, 51(6):3129-3138). Pressureat the base of the column was verified by TONOPEN® (Reichert, Depew,N.Y.).

Ca²⁺ Imaging

Flow-induced Ca²⁺ signals were examined in the presence and absence ofcilia in HTM cells. To remove primary cilia, 2 mM aqueous chlorohydrate(Sigma, St. Louis) was added for 48 hours. Prior to flow experiments,cells were washed three times with PBS, and fresh medium was added for24 hours. The Fura-2 loaded HTM cells were subjected to laminar flowrates as indicated. Single-cell analysis of the Ca²⁺ signals (ratio340/380 nm) of the same experiment was obtained. Intracellular Ca²⁺changes were monitored by ratiometric imaging of Fura-2 AM fluorescencein the HTM cells. HTM cells were loaded with Fura-2 AM for 1 hour at 37°C. in PBS with 40 μM fura-2 AM, 0.33 mM pyruvate, 0.901 mM Ca²⁺, 0.5 mMMg²⁺, 5.5 mM glucose, and 0.1% BSA. The Fura-2 AM-loaded HTM cells werewashed in PBS and were mounted in the chamber. Ca²⁺ imaging experimentswere performed using a monochromator-based TILL PHOTONICS imagingsystem, which was attached to an inverted ZEISS microscope equipped(×2.5 objective) with a back-illuminated ANDOR CCD camera. Fura-2 AM wasalternately excited at 345 nm and 380 nm and the emitted light wascollected using a 510-nm long-pass filter. The TILLVISION software (TILLPHOTONICS) was used to acquire all fluorescence imaging experiments andto perform the analysis of images. Images were acquired every 2 seconds.The background fluorescence was subtracted and the intracellular Ca²⁺concentration ([Ca²⁺]i) was calculated using the equation:[Ca²⁺]i=Kd×[(R−Rmin)/(Rmax−R)]×[Fmax(380)/Fmin(380)](R is thefluorescence ratio, Rmax is the ratio at saturation calciumconcentration, Rmin is the ratio at zero calcium in the medium,Fmin(380) is the fluorescence at 380 nm at zero Ca²⁺ concentration inthe medium, and Fmax(380) is the fluorescence at 380 nm at saturationCa²⁺ concentration) (Touw et al., Am. J. Physiol. Gastro. LiverPhysiology 2012, 302(1):G66-76).

Electron Microscopy

With an IRB approved study by Indiana University, human trabeculartissues were obtained at the time of trabeculectomy and immediatelyplaced in 2.5% Glutaraldehyde, 2% paraformaldehyde in 0.1 M NaCacodylate, pH 7.4. Images were obtained as described in Blitzer et al.(Proc. Natl. Acad. Sci. USA 2011, 108(7):2819-2824).

Lowe Syndrome Keratinocytes

A keratinocyte cell line was established from an 8-year old male patientpreviously diagnosed with Lowe syndrome with bilateral congenitalglaucoma and cataracts at Riley Children's Hospital, Indianapolis, Ind.after informed consent was obtained from the patient's parents and inaccordance with an IRB approved study by Indiana University. At 12-hoursafter birth, the patient was identified as having bilateral elevatedpressure, opaque corneas, and cataracts. Two weeks after delivery thepatient underwent left eye trabeculotomy and trabeculectomy surgeriesfor glaucoma, followed by the right eye. The patient then had cataractsurgery subsequently in both eyes. Urinalysis revealed proteinuria andtissue biopsy confirmed Lowe syndrome. The patient subsequently requiredmultiple eye surgeries for TOP lowering, resulting in counting fingervision in the right eye and hand-motion vision in the left eye.

Patient Mutation Analyses

With IRB approval, patient keratinocyte samples were used for DNAisolation. The 25 coding exons and cDNA ORF of OCRL were amplified understandard conditions and directly sequenced using an ABI 377 DNAsequencer (Applied Biosystems). Mutations were verified in an ethnicallymatched control individual.

Lowe Syndrome Patient Fibroblasts

Primary fibroblasts (GM01676 and GM03265, referred to as Lowe 1 and Lowe2, respectively) generated from two Lowe syndrome patients with defectsin the OCRL gene were obtained from the Coriell Institute for MedicalResearch (Camden, N.J.) and previously characterized by Luo et al. (Hum.Mol. Genet. 2012, 21(15):3333-3344) and Coon et al. (Hum. Mol. Genet.2012, 21(8):1835-1847). GM 01676 cells contain a mutation in the OCRLgene (2479C>T) resulting in a premature stop at codon 827 (R827X).

Statistical Analysis

Results are expressed as mean values±standard deviation (SD).Statistical analysis was performed using student t-tests (SPSS, Chicago,Ill.). A p-value of less than 0.05 was considered statisticallysignificant.

Example 1

In this Example, isolated trabecular meshwork (TM) tissue from theanterior segment of human eyes was analyzed for the presence of cilia byimmunofluorescence and electron microscopy.

Trabecular meshwork removed from human donor eyes and surgical TMspecimens during glaucoma surgery were immunostained with anti-Arl13b,acetylated α-tubulin (AcTub), γ-tubulin (γ-Tub), and DAPI (to visualizecell nuclei). TM sections of human eyes were fixed, sectioned andelectron microscopy performed. TEM images showed basal body and axonemalstructures (FIG. 1, arrows).

Trabecular meshwork (TM) tissue from the anterior segment of the eyecontains cilia-like structures (see schematic illustration in FIG. 1).As shown in FIGS. 2A and 2B, primary cilia were detected in TM tissuesby immunostaining for acetylated α-tubulin or Arl13b, a small GTPaselocalized in the cilia, while the basal body was detected by stainingfor γ-tubulin. Collectively, this revealed numerous cilia-likestructures in the uveoscleral region of the TM (FIG. 2A). The similarstaining of cilia in TM tissues that were removed en face immediatelyfollowing trabeculectomy from the eyes of glaucoma patients indicatedthat positive staining was not an artifact of fixation (FIG. 2B). Thedetection of primary sensory cilia with characteristic “9+0” structuresby electron microscopy confirmed their presence in the TM (FIGS. 2C and2D). Specificity for TM was verified by immunostaining of myocilin, aTM-enriched protein (FIG. 2E). Similar observations were noted in thetrabecular meshwork of murine (FIG. 2F), porcine (FIGS. 2G and 2H) andbovine (FIGS. 2I and 2J) eyes. These results demonstrated that primarycilia are present in the TMs of many mammalian species.

Serum starvation is a well-described method for the induction of primarycilia. To determine whether serum starvation could induce the formationof cilia, cultured human trabecular meshwork (HTM) cells were subjectedto serum starvation for 48 hours. Following serum starvation, HTM cellswere immunostained to detect Arl13b, IFT88 (an intraflagellar transportprotein), IFT43, IFT57, adenylate cyclase III (a cilia marker), andγ-tubulin (FIG. 2K).

HTM cells were then serum starved for 24 hours, 48 hours and 72 hours.As visualized in FIG. 3A, cilia length increased over time during serumstarvation. Measurements of cilia demonstrated that the average lengthsof cilia increased following serum deprivation. At 24 hours the cilialengths were 2.2±0.7 μm; at 48 hours, 4.3±0.6 μm; and were 5.6±0.4 m at72 hours (FIG. 3B). As illustrated in FIG. 3C, ciliation rate alsoincreased over time. These results strongly confirmed the presence offunctional primary cilia in trabecular meshwork cells.

Example 2

In this Example, the effect of pressure changes on cilia length in TMcells was determined.

HTM cells were serum starved for 48 hours, cultured in a pressurechamber at 0 mmHg, 30 mmHg and 50 mmHg pressure for 0 hour, 1 hour and 3hours. After cell fixation, cilia were imaged by immunostaining foracetylated α-tubulin. As shown in FIG. 4A, cilia length was reduced bypressure. Average cilia lengths were 5.3±0.2 μm in the normal pressuregroups and 3.9±0.2 μm in the high pressure (30 mmHg and 50 mmHg) groups(FIG. 4B). Ciliary length reduction by pressure occurred in atime-dependent fashion (FIG. 4C). Changes in ciliary length were bothtime- and dose-dependent processes.

TNFα and TGFβ are both highly abundant in the aqueous humor of patientswith acute and chronic open angle glaucoma. To determine whether theexpression of TNFα was upregulated in response to pressure, itstranscript levels were measured by qRT-PCR under different pressures.

HTM cells were serum-starved for 48 hours and treated with saline,saline plus 50 mmHg pressure, chlorohydrate, and chlorohydrate plus 50mmHg pressure for 3 hours. Levels of mRNA of TNFα, TGFβ1 and Gli1 werethen measured. Results presented represent three independentexperiments. Serum-starved HTM cells were also placed under 50 mmHgpressure for 0, 10, 20, 40, 60 and 80 minutes and analyzed forexpression of TNFα and IL-33. Results presented represent threeindependent experiments.

Following 60 minutes of application of 50 mmHg pressure, which mimicsacute angle closure glaucoma, a robust elevation of TNFα transcriptionwas observed (FIG. 4D). The lack of changes in IL-33 transcriptindicated that the effects on TNFα are specific (FIG. 4E). Further,application of 50 mmHg pressure markedly elevated the transcript levelsof TNFα and TGFβ1, but had modest effects on Gli1, a factor in the SonicHedgehog pathway that requires primary cilia for signaling (FIG. 4D).The dependence of these effects on functional cilia was then determinedby testing their sensitivity to chlorohydrate, which effectively removescilia. Treatment of cells with chlorohydrate reduced the elevation ofgene transcription in response to pressure (FIG. 4D).

In a complementary approach, cilia were ablated by shRNA knockdown ofRab8, which inhibits ciliogenesis. Cells with reduced expression of Rab8also showed a reduction of gene transcription (Rab8 and OCRL) inresponse to pressure (FIGS. 4F-4H).

Together, these experiments strongly suggested that enhancedtranscription of TNFα and TGFβ1 by pressure occurs via a cilia-dependentprocess.

Example 3

To further characterize the distribution of ciliary proteins underelevated pressure conditions, intraflagellar transport (IFT) proteinswere examined.

The distribution of proteins involved in both anterograde (IFT57, IFT88)and retrograde (IFT20, IFT43, IFT144) ciliary trafficking were examinedusing immunohistology. Specifically, HTM cells were serum-starved toinduce ciliogenesis, followed by treatment with or without elevatedpressure (50 mmHg). Representative images of immunostaining withantibodies for IFT88 (green), acetylated α-tubulin (red), and DAPI(blue) show the loss of IFT88 in the distal tip of cilia under elevatedpressure conditions (FIGS. 5 and 6). Only IFT88 was found to have amarkedly altered distribution under increased pressure conditions. Incontrol HTM cells, TFT88 distributed to the base as well as to the tipof the axoneme; in HTM cells treated with pressure, IFT88 accumulated atthe base of the cilia with a marked decrease at the ciliary tip.

Thus, elevated pressure in cells alter IFT distribution with a resultantchange in ciliary protein trafficking.

Example 4

Glaucoma frequently develops after prolonged elevated intraocularpressure (Alward et al., N. Engl. J. Med. 1998, 338(15):1022-1027). Lowesyndrome is a rare X-linked disorder that presents with congenitalglaucoma and cataracts, as well as renal dysfunction and CNSabnormalities (Attree et al., Nature 1992, 358(6383):239-242). In thisExample, genetic and cellular analysis of a Lowe syndrome patient wasconducted.

An 8-year-old male patient with Lowe syndrome who was born withbilateral congenital glaucoma and cataracts was identified. Table 1summarizes the clinical profile of the patient.

TABLE 1 CLINICAL PROFILE OF LOWE SYNDROME PATIENT Right Eye Left EyeVision Counting fingers Hand motion Intraocular pressure 40 mmhg 55 mmhgAxial length 20.74 20.49 Diagnosis Cataract (discoid) Cataract (discoid)glaucoma glaucoma Band keratopathy Surgeries Lensectomy LensectomyTrabeculectomy Trabeculectomy trabeculotomy Trabeculotomy Ahmed shuntvalve Ecp

Less than 12 hours postpartum, the patient was noted to have cornealedema and elevated pressures in both eyes and increased optic nervecupping (FIG. 7, cup represented by dashed line). The patientsubsequently underwent multiple surgeries for glaucoma includingtrabeculectomy and trabeculotomy with poorly controlled disease.

DNA sequencing revealed a novel missense mutation in the region encodingthe 5-phosphatase domain of the OCRL gene (c.1661 A>C; p.D499A) (FIG.8A). A model of the OCRL structure was generated by PyMol, ID: 3MTC.pdb.Based on its position in the OCRL structure, this aspartic acid residuewas predicted to affect the 5-phosphatase activity of OCRL (FIG. 8B).

To further assess the role of OCRL in cilia, the localization of OCRLwas visualized in human TM cells following serum starvation. Normalcontrol (NHKC), Lowe syndrome patient (Lowe 3) and patient transducedwith WT-GFP-OCRL (Lowe 3+WT) keratinocytes were serum starved for 48hours and stained for acetylated α-tubulin and γ-tubulin.Immunofluorescence showed that OCRL was concentrated in the axoneme orthe membrane of the primary cilium (FIG. 8C). Consistent with OCRLfunctioning in the cilia of retinal pigment epithelial (RPE) cells, thecilia in keratinocytes derived from this patient were defective. Forinstance, following serum-starvation, these cells had shortened ciliaversus keratinocytes obtained from a healthy individual (FIG. 8D). Uponre-introduction of wildtype OCRL (Lowe 3+WT OCRL), the shortened ciliaphenotype was rescued.

OCRL mutant cDNA (OCRL D499A) was expressed in HTM cells to determineits effect on cilia formation. Expression of OCRL mutant cDNA resultedin the formation of significantly shorter cilia structures in thesecells upon serum-starvation as compared to control cells (FIGS. 8E-8G).

These data implicate OCRL in cilia formation in TM cells, which in turn,is likely required for pressure sensing in the eye.

Given the potential role of OCRL in pressure sensing, the requirementfor OCRL for the activation of transcription in HTM cells in response topressure was assessed.

The expression of OCRL was silenced in HTM cells with lentiviral shRNA.Ciliogenesis was then induced by serum starvation, followed by pressurestimulation (see, FIG. 4H and FIGS. 8H and 8I). Cells with reduced OCRLexhibited a significant loss of pressure-dependent TGFβ, TNFα, and Gli1transcriptional activity, which was restored by re-expression ofwildtype OCRL (FIG. 8J).

In a separate approach, pressure-dependent transcription of TGFβ wasassessed in primary fibroblasts obtained from wild-type control (NHF558cells) versus primary fibroblasts from two Lowe syndrome patients withnonfunctional OCRL protein (described in Luo et al., Hum. Mol. Genet.2012, 21(15):3333-3344). NHF558, Lowe 1 and Lowe 2 fibroblasts, andOCRL-WT expressing Lowe 1 and Lowe 2 fibroblasts were serum starved for48 hours and incubated with or without 50 mmHg pressure for 3 hours.TGFβ mRNA level was determined by RT-PCR. Pressure-induced TGFβ mRNAlevels, measured as a ratio of 50 mmHg fold change over control averagedover three independent experiments is shown (FIG. 8K).

The levels of TGFβ transcript modestly increased following elevatedpressure in mutant cells whereas cells expressing wildtype OCRL showeddramatically increased elevation of TGFβ in response to pressure (FIG.8K). This further supports the assertion that pressure activates thetranscription of TNFα and TGFβ in a manner that requires cilia, which inturn are dependent on functional OCRL.

Example 5

Transient receptor potential vanilloid 4 (TRPV4) is a mechanosensitivecalcium permeable cation channel that localizes to cilia where itfunctions in osmotic regulation. TRPV4 dysregulation has been implicatedin diseases with fluid overload, such as hydrocephalus and heartfailure. In this Example, the role of TRPV4 in TM cilia and respondingto mechanosensory signals was determined.

HTM cells were serum starved for 48 hours and immunostained withanti-TRPV4 and anti-acetylated α-tubulin antibodies. Nuclei werevisualized by DAPI staining. Additionally, HEK293 cells were transfectedwith FLAG-TRPV4, GFP-OCRL, and GFP alone. Immunoblots for OCRL (IB:OCRL)and FLAG-TRPV4 were performed after immunoprecipitation for FLAG.

As shown in FIGS. 9A and 9E, both OCRL and TRPV4 were expressed in HTMcells and co-localized in the primary cilia as indicated by staining foracetylated tubulin. TRPV4 expression was similar in Lowe patientfibroblasts and normal fibroblasts (FIGS. 9B and 9C). Consistent withtheir colocalization, exogenously expressed TRPV4 specificallyco-immunoprecipitated with OCRL (FIG. 9D).

To determine if the D499A OCRL mutant has altered localization andinteraction with TRPV4, the localization and interaction with TRPV4 ofwildtype OCRL or mutant D499A OCRL in TM cells was determined. HTM cellswere serum starved and transfected with FLAG-TRPV4, GFP-OCRL andGFP-D499A-OCRL, followed by immunostaining with anti-acetylatedα-tubulin and anti-FLAG antibodies. Nuclei were visualized by DAPIstaining. Immunoblots for OCRL and FLAG-TRPV4 after immunoprecipitationwas performed on HEK293 cells transfected with FLAG-TRPV4, GFP-OCRL andGFP-D499A-OCRL.

While TRPV4 localized in the primary cilia in cells expressing wild-typeOCRL, its localization to cilia was markedly diminished in cellsexpressing the D499A OCRL mutant (FIG. 9E). The D499A OCRL mutantimmunoprecipitated with TRPV4 to a much greater degree than withwildtype OCRL (FIG. 9F). These results demonstrate that the D499A has adominant-negative effect of sequestering TRPV4 out of the cilia.

Further, the function of the OCRL D499A mutant was assessed in vitro,and it was found that the enzymatic activity of PI(4,5)P2 5-phosphatasewas decreased twofold compared with the wild-type enzyme (FIG. 10A).However, the stability of the protein was not significantly affected(FIGS. 10B & 10C). Taking these data together, OCRL is shown to interactwith TRPV4 calcium channel and both proteins localize within the primarycilia.

Example 6

In this Example, the dependence of TRPV4 on OCRL for responding tomechanosensory signals to transport Ca²⁺ was analyzed.

For calcium flow experiments, HTM cells and NHF cells were firstincubated in 3% BSA in PBS and stained with Fura2 dye for 1 hour. Ca²⁺flux was induced by laminar flow gradient (16 μl/s) over the cellsurface, which bends cilia, followed by 10 mM KCl. Cells were imaged forcalcium during flow. Calcium flux was also measured by F340/380 ratio inHTM cells and NHF cells after treatment with the 0.1 μM TRPV4 agonistGSK1016790A. Results presented represent calcium mobilization in threeindependent experiments.

In Fura2-loaded HTM cells and NHF cells, Ca²⁺ flux was induced bylaminar flow gradient (FIG. 11A). Calcium flux as measured by F340/380ratio in NHF cells was also elicited by treatment with the TRPV4 agonistGSK1016790A (FIG. 11B). However, this TRPV4 agonist failed to elicitcalcium mobilization in Lowe patient fibroblasts.

HTM cells treated, with or without pressure, were further evaluated todetermine their response to TRPV4 agonist. HTM cells were serum-starvedfor 48 hours to induce ciliogenesis, followed by treatment with 0 or 50mmHg for 3 hours. Cells were loaded with Fura-2 AM dye for 1 hour,followed with 0.1 M TRPV4 agonist GSK1016790A. The ratio of F340/380(value X 1000) that indicates calcium mobilization is shown in FIG. 12A.Particularly, in ciliated HTM cells treated with 50 mmHg hydrostaticpressure, a significant decrease in TRPV4-induced calcium flux wasobserved.

OCRL is required for TRPV4-mediated calcium signalling in HTM cells. HTMcells were additionally treated with OCRL siRNA or scrambled controlsiRNA, with or without wild-type OCRL or OCRL-D499A rescue. 0.1 μM TRPV4agonist GSK1016790A treatment was performed and calcium mobilization byF340/380 (value X 1000) was measured and shown. As shown in FIG. 12B, adecrease in F340/F380 fura-2 signaling was observed using OCRL siRNA orscrambled control siRNA-treated HTM cells in the presence ofTRPV4-agonist, which was rescued by wild-type OCRL, but not by the D499Amutant.

Additionally, a Lowe syndrome patient's keratinocytes were isolated andassayed for their response to TRPV4 treatment. NHKC or Lowekeratinocytes were serum-starved, loaded with Fura-2 AM and then treatedwith 0.1 μM TRPV4 agonist GSK1016790A. F340/380 ratio (value X 1000) wasdetermined. The treatment with TRPV4 agonist failed to elicit calciummobilization in Lowe patient keratinocytes compared with normalkeratinocytes (FIG. 12C).

Based on the results presented above, the functional consequences ofactivating TRPV4 on eye pressure were therefore assessed in the wpk ratmodel. The wpk rat is a well-established model for studying ciliopathy,as these animals develop hydrocephalus that responds to cilia-basedtherapy.

8-day old wpk^(−/−) rats were treated with sham, TRPV4 agonist (50 ng/g,GSK 1016790A), or antagonist (HC 067047) once per day for 8 days. At day17, intraocular pressure measurements (in mmHg) were performed using aTonolab tonometer 24 hours after treatment.

In 17-day-old wpk^(−/−) rats, intraocular pressure was significantlyreduced with systemic GSK1016790A treatment but not by sham treatment orwith a TRPV4 antagonist (HC 067047) (FIG. 11C).

This finding was confirmed in wild-type C5BL/6 mice treated withsystemic TRPV4 agonist, antagonist, or sham control, daily over 4 days.Nine-week old C57BL/6 WT mice were treated with sham, TRPV/4 agonist GSK1016790A or antagonist HC 067047 for 4 days. IOP was measured using aTonolab tonometer 24 hours after treatment. As shown in FIG. 12Darticle, a decrease in IOP from 12.4 to 10.3 mmHg was seen.

Finally, IOP was assessed in 7-month old TRPV4^(−/−) versus TRPV^(4−/−)mice. IOP in TRPV4^(−/−) mice was shown to be elevated compared withcontrol TRPV4^(−/−) mice (FIG. 12E). The primary cilia in the TM ofthese animals were evaluated by immunofluorescence staining for ciliarymarkers with Arl13b and γ-tubulin; the cilia were found to be indeedshorter in the TRPV4^(−/−) mice (FIGS. 12F & 12G). These resultsindicated that functional OCRL is necessary for TRPV4 distribution,which in turn is required for calcium signaling and cilia growth inresponse to mechanical stimuli.

Example 7

In this Example, additional TRPV4 agonists and antagonists wereanalyzed.

Both agonists and antagonists were diluted in PBS to indicatedconcentrations and given i.p. to animals as shown in FIGS. 13A & 13B.IOP measurements were performed with TONOLAB®. As shown in FIGS. 13A &13B, only TRPV4 agonists have an IOP lowering effect.

Example 8

In this Example, a time-dependent analysis using TRPV4 agonists areconducted.

Both agonists and antagonists were diluted in PBS to indicatedconcentrations and given i.p. to animals as shown in FIGS. 14A & 14B.IOP measurements were performed with TONOLAB®. As shown in FIGS. 14A &14B, IOP-lowering effect is increased within 45 minutes ofadministration of the TRPV4 agonists.

Example 9

In this Example, TRPV4 distribution in human trabecular meshwork (TM)cilia is analyzed.

Cilia counting was performed on the fluorescent images. Student t-testwas used to compare the two groups. As shown in FIG. 15, the TRPV4distribution in human TM cilia support its function as a mechanosensorin the eye. Based on the results, TRPV4 trafficking to the cilia is anessential step for the release of intracellular calcium, which may leadto subsequent enhanced Ca²⁺/calmodulin-dependent protein kinase Hactivity and up-regulate endothelial nitric oxide synthase. Nitric oxidehas been shown as an effector path-way for lowering intraocularpressure. Accordingly, it is believed that the primary cilia within TMcells may serve as an afferent pathway for signal transduction.

Mechanosensation of pressure underlies a number of important humandiseases including the development of hypertension and glaucoma.Defective sensation of pressure may result in imbalance of aqueoushumor, resulting in elevated intraocular pressure. Low levels of eyepressure result in structural changes of the retina and poor vision,while elevated eye pressure may damage the optic nerve. As illustratedin the schematic presented in FIG. 16, a role of primary cilia in thesensation of pressure in human TM is presented. The presence of ciliathat shortened in response to pressure in TM cells was identified andproper cilia function was determined to be essential for pressuresensation in these cells. These effects were found to require both OCRLand TRPV4 in a manner where OCRL contributed to proper localization andfunction of TRPV4 in the cilia. This, in turn, is necessary for calciumflux that regulates the transcriptional programs that coordinate ciliafunction with pressure sensing. As provided herein, cilia in the TMcells regulated pressure of the eye, which, when dysregulated, isstrongly implicated in the pathogenesis of glaucoma.

These results surprisingly allow for the treatment of intraocularpressure and, in particular, glaucoma. The present disclosure has abroad and significant impact, as it allows for treating glaucoma anddysregulation of eye pressure that can lead to vision loss andblindness.

1.-23. (canceled)
 24. A method for reducing intraocular pressure in anindividual in need thereof, the method comprising: administering acomposition comprising a transient receptor potential cation channelsubfamily V member 4 (TRPV4) agonist to the individual, wherein theTRPV4 agonist is selected from the group consisting of3-oxohexahydro-1H-azepin, azepine and acyclic 1,3-diamine andderivatives of these compounds.
 25. The method of claim 24, wherein theTRPV4 agonist is selected from the group consisting ofN-{(1S)-1-[({(4R)-1-[(4-chlorophenyl)sulfonyl]-3-oxohexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl}-1-benzothiophene-2-carboxamide;N-{(1S)-1-[({(4R)-1-[(4-fluorophenyl)sulfonyl]-3-oxohexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl}-1-benzothiophene-2-carboxamide;N-{(1S)-1-[({(4R)-1-[(2-cyanophenyl)sulfonyl]-3-oxohexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl}-1-methyl-1H-indole-2-carboxamide;N-{(1S)-1-[({(4R)-1-[(2-cyanophenyl)sulfonyl]hexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl-1-methyl-1H-indole-2-carboxamide;N-{(1S)-1-[((3-[[(cyanophenyl)sulfonyl](methyl)amino]propyl}amino)carbonyl]-3-methylbutyl}-1-benzothiophene-2-carboxamide;andN-{(1S)-1-[({3-[[(2,4-dichlorophenyl)sulfonyl](methyl)amino]propyl}amino)carbonyl]-3-methylbutyl}-1-benzothiophene-2-carboxamide.26. The method of claim 24, wherein the individual is administered fromabout 50 ng/g body weight to about 500 ng/g body weight of the TRPV4agonist.
 27. The method of claim 24, wherein the individual isadministered the TRPV4 agonist using a method selected from the groupconsisting of topically, periocularly, intraocularly, and combinationsthereof.
 28. The method of claim 27, wherein the individual isadministered the TRPV4 agonist using intraocular injection.
 29. A methodfor treating glaucoma in an individual in need thereof, the methodcomprising: administering a composition comprising a transient receptorpotential cation channel subfamily V member 4 (TRPV4) agonist to theindividual, wherein the TRPV4 agonist is selected from the groupconsisting of 3-oxohexahydro-1H-azepin, azepine and acyclic 1,3-diamineand derivatives of these compounds.
 30. The method of claim 29, whereinthe TRPV4 agonist is selected from the group consisting ofN-{(1S)-1-[({(4R)-1-[(4-chlorophenyl)sulfonyl]-3-oxohexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl}-1-benzothiophene-2-carboxamide;N-{(1S)-1-[({(4R)-1-[(4-fluorophenyl)sulfonyl]-3-oxohexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl}-1-benzothiophene-2-carboxamide;N-{(1S)-1-[({(4R)-1-[(2-cyanophenyl)sulfonyl]-3-oxohexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl}-1-methyl-1H-indole-2-carboxamide;N-{(1S)-1-[({(4R)-1-[(2-cyanophenyl)sulfonyl]hexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl}-1-methyl-1H-indole-2-carboxamide;N-{(1)-1-[((3-[[(cyanophenyl)sulfonyl](methyl)amino]propyl}amino)carbonyl]-3-methylbutyl-1-benzothiophene-2-carboxamide;andN-{(1S)-1-[({3-[[(2,4-dichlorophenyl)sulfonyl](methyl)amino]propyl}amino)carbonyl]-3-methylbutyl}-1-benzothiophene-2-carboxamide.31. The method of claim 29, wherein the individual is administered fromabout 50 ng/g body weight to about 500 ng/g body weight of the TRPV4agonist.
 32. The method of claim 29, wherein the individual isadministered the TRPV4 agonist using a method selected from the groupconsisting of topically, periocularly, intraocularly, and combinationsthereof.
 33. The method of claim 32, wherein the individual isadministered the TRPV4 agonist using intraocular injection.
 34. A methodfor treating a ciliopathy in an individual in need thereof, the methodcomprising: administering a composition comprising a TRPV4 agonist tothe individual, wherein the TRPV4 agonist is selected from the groupconsisting of 3-oxohexahydro-1H-azepin, azepine and acyclic 1,3-diamineand derivatives of these compounds.
 35. The method of claim 34, whereinthe TRPV4 agonist is selected from the group consisting ofN-{(1S)-1-[({(4R)-1-[(4-chlorophenyl)sulfonyl]-3-oxohexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl}-1-benzothiophene-2-carboxamide;N-{(1S)-1-[({(4R)-1-[(4-fluorophenyl)sulfonyl]-3-oxohexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl}-1-benzothiophene-2-carboxamide;N-{(1S)-1-[({(4R)-1-[(2-cyanophenyl)sulfonyl]-3-oxohexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl}-1-methyl-1H-indole-2-carboxamide;N-{(1S)-1-[({(4R)-1-[(2-cyanophenyl)sulfonyl]hexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl}-1-methyl-1H-indole-2-carboxamide;N-{(1)-1-[((3-[[(cyanophenyl)sulfonyl](methyl)amino]propyl}amino)carbonyl]-3-methylbutyl-1-benzothiophene-2-carboxamide;andN-{(1S)-1-[({3-[[(2,4-dichlorophenyl)sulfonyl](methyl)amino]propyl}amino)carbonyl]-3-methylbutyl}-1-benzothiophene-2-carboxamide.36. The method of claim 34, wherein the individual is administered fromabout 50 ng/g body weight to about 500 ng/g body weight of the TRPV4agonist.
 37. The method of claim 34, wherein the individual isadministered the TRPV4 agonist using a method selected from the groupconsisting of topically, periocularly, intraocularly, and combinationsthereof.
 38. The method of claim 40, wherein the individual isadministered the TRPV4 agonist using intraocular injection.
 39. Themethod of claim 34, wherein the ciliopathy is selected from the groupconsisting of retinitis pigmentosa, renal cysts, polydactyly, anddevelopmental delays.